Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics

Abstract

Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling.

Recapitulation of molecular regulators of nuclear motion during cell migration

“…During cell migration, actin regulating proteins control F-actin formation so that nuclear movement is coordinated. Refilin proteins including RefilinA and RefilinB are a novel family of filamin-binding shortlived actin regulators involved in cellular phenotypic alterations such as epithelial-to-mesenchymal transition (EMT) which makes cells to promote metastasis by decreasing nuclear stiffness that is induced by the loss of lamin A/C and allows nucleus to translocate to the foreign microenvironment with severe physical stress [18,[41][42][43]. RefilinA promotes actin-binding filamin A (FLNA) to assemble F-actin bundles whereas RefilinB organizes a perinuclear actin cap [44].…”

“…Nuclear molecules involved in cell migration. of filamin-binding short-lived actin regulators that are involved in cellular phenotypic alterations such as epithelialto-mesenchymal transition[41,44] Refilin A: promotes the actin-binding filamin A (FLNA) to convert FLNA into an F-actin bundles Refilin B: organizes a perinuclear actin cap Filamin Cytoplasm A downstream effector of the refilin proteins, coordinates the reorganization of perinuclear actin cytoskeleton and regulates nuclear motion…”

The filamin-B–refilin axis – spatiotemporal regulators of the actin-cytoskeleton in development and disease

“…The short half-life of the refilins is related to them being mostly unstructured ( Fig. 2B) and the presence of several destruction motifs (Gay et al, 2016). In cells, the stability of intrinsically unstructured proteins (IUPs) is finely tuned so they persist in an appropriate stoichiometry with their physiological targets (Dyson and Wright, 2005;Gsponer et al, 2008).…”

“…In the case of refilins, their stabilization depends on interaction with their target protein, that is, filamin. In the human M2 cell line that does not express filamin, ectopically expressed rat refilin-A and refilin-B are immediately degraded (Gay et al, 2016). In this context, co-expression with filamin or filamin minigenes encompassing their refilin-binding domains, results in stabilization of refilins such that they have a half-life of between 30 min and 4 h depending on the cell culture density (Gay et al, 2016).…”

“…When refilin is bound to filamin, its degradation relies on complex degradation pathways that recognize several destruction motifs within the N-terminal and C-terminal domains (Gay et al, 2016). The N-terminal dimerization domain of refilins harbors two (A) A schematic of a vertebrate filamin dimer.…”